Abstract

Progesterone is an effective anticonvulsant against pentylenetetrazol (PTZ) seizures. This action is hypothesized to require the metabolic conversion of progesterone to the γ-aminobutyric acidA receptor potentiating neuroactive steroid allopregnanolone by 5α-reductase isoenzymes followed by 3α-hydroxy oxidoreduction. We evaluated this possibility using the competitive 5α-reductase inhibitor finasteride. Progesterone (50–200 mg/kg, i.p.) protected mice against PTZ-induced seizures in a dose-dependent manner (ED50, 94 mg/kg). Pretreatment with finasteride (50–300 mg/kg, i.p.) produced a dose-dependent (ED50, 146 mg/kg) reversal of the protective effects of progesterone (2 × ED50 dose = 188 mg/kg). In contrast, finasteride (up to 300 mg/kg) failed to affect the anticonvulsant activity of allopregnanolone (10–30 mg/kg, i.p.; ED50, 12 mg/kg). Finasteride (up to 300 mg/kg) did not block the protective effect of high doses of progesterone (250–350 mg/kg) on tonic hindlimb extension in the maximal electroshock seizure test (progesterone ED50, 235 mg/kg). The anticonvulsant activity of progesterone against PTZ-induced seizures can be blocked by 5α-reductase inhibition, providing strong evidence that the anticonvulsant effect of the steroid in this model is mediated by its active metabolite allopregnanolone.

The steroid hormone progesterone is well known to have potent anticonvulsant and sedative-hypnotic actions in animals and humans (Selye, 1942; Spiegel and Wycis, 1945; Merryman et al., 1954; Craig, 1966). These effects of progesterone are hypothesized to be a consequence of its metabolic conversion to the endogenous neuroactive steroids allopregnanolone (5α-pregnan-3α-ol-20-one) and pregnanolone (5β-pregnan-3α-ol-20-one) (Atkinson et al., 1965;Conney et al., 1966; Gyermek et al., 1967, 1968). In contrast to other steroid actions that are mediated by effects on gene transcription, neuroactive steroids rapidly alter the excitability of neurons by directly modulating the activity of neuronal membrane ion channels, including the γ-aminobutyric acidA(GABAA) receptor Cl− ion channel complex (Majewska, 1992; Paul and Purdy, 1992; Gee et al., 1995). Allopregnanolone and pregnanolone produce a powerful enhancement of GABAA receptor responses in vitro (Majewska et al., 1986; Harrison et al., 1987; Gee et al., 1988; Peters et al., 1988) and, like other potentiators of GABAAreceptor responses, show potent anticonvulsant, anxiolytic, and sedative activities when administered in vivo (Belelli et al., 1989;Bitran et al., 1991; Wieland et al., 1991; Kokate et al., 1994). Although it has been speculated that the anticonvulsant effects of progesterone are due to its neuroactive steroid metabolites, there is no direct evidence supporting this hypothesis. In the present study we sought to establish that the anticonvulsant effects of progesterone are due to its metabolic conversion to a 5α-reduced metabolite by 4-ene-3-oxosteroid 5α-oxidoreductase (5α-reductase) isoenzymes (Fig. 1). We examined the effect of the 4-azasteroid competitive 5α-reductase inhibitor finasteride (Rittmaster, 1997) on the anticonvulsant activity of progesterone in the mouse pentylenetetrazol (PTZ) and maximal electroshock (MES) seizure tests. Our results provide strong support for the conclusion that progesterone requires activation by 5α-reduction in order to have anticonvulsant activity in the PTZ test (but not the MES test).

Metabolic pathway for conversion of progesterone to allopregnanolone (5α,3α-pregnanolone) and its blockade by finasteride, a 5α-reductase inhibitor.

Materials and Methods

Animals.

Male NIH Swiss mice (25–30 g) were obtained from the National Institutes of Health (NIH) animal program. Animals were allowed to acclimatize with free access to food and water for a 24-h period before testing. All procedures were carried out under strict compliance with the NIH Guide for the Care and Use of Laboratory Animals under a protocol approved by the NIH Animal Use Committee.

PTZ Seizure Test.

Steroids were evaluated for protective activity against PTZ-induced clonic seizures according to the procedure described by White et al. (1995). In brief, mice were injected i.p. with the steroid and 15 min (allopregnanolone) or 30 min (progesterone) later (or at the specified intervals in the time course studies) received a s.c. injection of PTZ (85 mg/kg). Animals were then observed for a 60-min period. Mice failing to show clonic spasms lasting longer than 5 s were scored as protected. The interval between allopregnanolone administration and PTZ injection was the time of peak effect as determined previously (Kokate et al., 1994). Finasteride was administered i.p. 1 min before injection of progesterone or allopregnanolone. Finasteride by itself at doses as high as 300 mg/kg failed to produce any protective effect against clonic seizures induced by PTZ.

MES Seizure Test.

Animals were subjected to a 0.2-s, 60-Hz electrical stimulus through corneal electrodes (5-mm diameter stainless steel balls) wetted with 0.9% saline. The electroshock unit was adjusted to deliver a current of 50 mA. Animals failing to show tonic hindlimb extension were scored as protected. Progesterone was injected i.p. 30 min before the MES seizure test. Finasteride was administered i.p. 1 min before the progesterone injection.

Drug Solutions.

Progesterone, allopregnanolone, and finasteride solutions were made fresh daily in aqueous 30% hydroxypropyl-β-cyclodextrin (β-cyclodextrin; Research Biochemicals, Natick, MA). Further dilutions were made using 0.9% saline. Drug solutions were administered in a volume equaling 1% of the animal’s body weight. All drugs were obtained from Sigma Chemical Co. (St. Louis, MO).

Data Analysis.

To construct dose-effect curves, progesterone or allopregnanolone were tested at several doses spanning the dose producing 50% protection (ED50). At least eight mice were tested at each dose. ED50 values and the corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4.2, MicroComputer Specialists, Philadelphia, PA). A similar analysis was used for determination of the CD50dose of PTZ (the dose at which 50% of tested animals exhibited convulsions). Dose-response data were fit to the logistic function 100/[1 + (ED50/x)nH] where x is the dose administered, andnH is an empirical parameter describing the steepness of fit.

Results

Anticonvulsant Activity of Progesterone.

As shown in Fig.2, progesterone (50–200 mg/kg) protected mice against PTZ-induced seizures in a dose-dependent fashion. The ED50 value obtained from these data was 94 mg/kg (95% CL: 81–108). In vehicle control experiments, 30% β-cyclodextrin failed to protect any of 16 animals tested against PTZinduced seizures. We also determined the time course for protection against PTZ-induced seizures using a dose of progesterone that produced complete protection at 30 min (200 mg/kg). As shown in Fig.3, the steroid produced a sustained anticonvulsant effect for 2 h. The anticonvulsant activity wore off during the subsequent 2-h period.

Time course for protection against PTZ-induced seizures by 200 mg/kg progesterone. Each point represents data from 8 mice.

Effect of Finasteride on Anticonvulsant Activity of Progesterone.

In a preliminary study, we examined the effects of 100 and 200 mg/kg finasteride on the anticonvulsant activity of 100 mg/kg progesterone in the PTZ seizure test. As shown in Fig.4, 100 mg/kg finasteride produced a moderate inhibition of the anticonvulsant activity of progesterone. Increasing the dose of finasteride to 200 mg/kg almost completely blocked the anticonvulsant effects of progesterone. In further experiments, the partial inhibition of the anticonvulsant activity of progesterone produced by 100 mg/kg finsasteride was overcome by increasing the dose of progesterone to 250 mg/kg (8 of 8 animals protected). Similarly, the near-complete inhibition produced by 200 mg/kg finasteride was partially overcome by 250 mg/kg progesterone (5 of 8 animals protected). We next determined the dose-response relationship for finasteride inhibition of the anticonvulsant activity of a dose of progesterone (2 × ED50 = 188 mg/kg) that produced complete protection against PTZ-induced seizures in all 16 control animals tested. Pretreatment with finasteride produced a dose-dependent inhibition of the anticonvulsant activity of progesterone. At doses of finasteride >200 mg/kg, there was complete inhibition of the anticonvulsant activity of the steroid (Fig.5). The ED50 value for finasteride inhibition of the anticonvulsant activity of progesterone was 146 mg/kg (95% CL: 125–170).

Dose-response relationship for finasteride inhibition of the anticonvulsant activity of progesterone (188 mg/kg) in the PTZ test. Finasteride (50–300 mg/kg) was administered 1 min before the administration of progesterone. Each point represents data from 8 to 16 mice.

To ascertain whether finasteride itself affects PTZ seizure threshold, we determined the dose-response relationships for PTZ (30–80 mg/kg) 30 min after animals were injected with vehicle (30% β-cyclodextrin) or 200 mg/kg finasteride. As illustrated in Fig.6, the dose-response relationships were overlapping, indicating that finasteride is neither proconvulsant or anticonvulsant. The CD50 values for the vehicle control and finasteride pretreatment groups were 51 mg/kg (95% CL: 45–59) and 52 mg/kg (95% CL: 44–61), respectively.

Dose-response relationships for seizure induction by PTZ (30–80 mg/kg, s.c.) in vehicle control (30% β-cyclodextrin) and finasteride-pretreated (200 mg/kg) mice. The pretreatment was administered 30 min before the administration of PTZ. Each point represents data from 8 to 10 mice.

Lack of Effect of Finasteride on Anticonvulsant Activity of Allopregnanolone.

The progesterone metabolite allopregnanolone (3–50 mg/kg, i.p.) produced a dose-dependent inhibition of PTZ-induced seizures with complete protection at doses ≥ 30 mg/kg (Fig.7A). The ED50 value for allopregnanolone obtained from this dose-response relationship was 12 mg/kg (95% CL: 10–16). Pretreatment with 200 mg/kg finasteride produced little or no effect on the anticonvulsant activity of 10, 15, and 30 mg/kg allopregnanolone (Fig. 7B). In an additional experiment, 300 mg/kg finasteride did not alter the complete protection produced by 30 mg/kg allopregnanolone (8 animals tested).

A, anticonvulsant activity of allopregnanolone (3–50 mg/kg, i.p.) in the PTZ seizure test. Allopregnanolone was administered 15 min before the injection of PTZ. Each point represents data from 10 mice. B, lack of effect of finasteride (200 mg/kg) on the anticonvulsant activity of allopregnanolone (10, 15, and 30 mg/kg). Finasteride was administered 1 min before administration of allopregnanolone. Each bar represents data from 10 mice.

Effects of Progesterone and Allopregnanolone in MES Seizure Test.

Progesterone in high doses (200–350 mg/kg) blocked the tonic hindlimb extension elicited by electroshock in the MES test. As illustrated in Fig. 8A, the effect of progesterone occurred in a dose-dependent fashion, and at the highest dose tested there was complete protection. The ED50 value obtained from these data was 235 mg/kg (95% CL: 212–263). Allopregnanolone was not effective in the MES test at doses of 30 and 50 mg/kg (8 animals tested at each dose). At 100 mg/kg, 3 of 16 animals tested were protected. Because doses higher than 100 mg/kg could not be tested due to solubility limitations, we could not assess whether allopregnanolone would have complete protective activity in the MES test similar to progesterone at very high doses.

A, anticonvulsant activity of progesterone (150–350 mg/kg, i.p.) in the MES seizure test. The MES test was performed 30 min after the progesterone injection. Each point represents data from 8 to 16 mice. B, lack of effect of finasteride on the anticonvulsant activity of progesterone (350 mg/kg, i.p.) in the MES test. Each bar represents data from 8 to 16 animals.

Lack of Effect of Finasteride on Anticonvulsant Activity of Progesterone in MES Test.

Finasteride (100–300 mg/kg) did not block the anticonvulsant effect of a high dose of progesterone (350 mg/kg) in the MES seizure test (Fig. 8B). Similarly, in additional experiments, 100 and 200 mg/kg finasteride failed to inhibit the anticonvulsant activity of a lower dose of progesterone (250 mg/kg). This lower dose of progesterone protected 9 of 16 naive animals and 5 of 8 finasteride-treated (100 or 200 mg/kg) animals. In vehicle control experiments, 30% β-cyclodextrin failed to protect any of 16 animals from tonic hindlimb extension. Furthermore, 100 mg/kg finasteride by itself did not block the tonic hindlimb extension due to MES in any of 16 animals tested. However, higher doses of finasteride alone exhibited partial protective activity: 3 of 16 animals protected at 200 mg/kg, and 8 of 16 animals protected at 300 mg/kg.

Discussion

In this study we show for the first time that the protective activity of progesterone in the PTZ seizure test can be blocked by the 5α-reductase inhibitor finasteride. Our results are consistent with two recent studies demonstrating that the anxiolytic (Bitran et al., 1995) and anesthetic (Korneyev and Costa, 1996) effects of progesterone can also be attenuated by 5α-reductase inhibition.

As observed previously by others (Selye, 1942; Craig, 1966), progesterone exhibited potent anticonvulsant activity against PTZ-induced seizures. This anticonvulsant activity was more prolonged (Fig. 3) than the anticonvulsant effect of the progesterone metabolite allopregnanolone (see Fig. 7 in Kokate et al., 1994), consistent with the possibility that progesterone can act as a depot for an active metabolite. Indeed, the anticonvulsant effect of progesterone in the PTZ test was blocked by finasteride in a dose-dependent manner, indicating that it must be activated by 5α-reduction. A critical control to eliminate the possibility of nonspecific effects of finasteride was the determination of whether the enzyme inhibitor affects the anticonvulsant activity of the progesterone metabolite allopregnanolone. Because the inhibitor failed to alter the anticonvulsant activity of the metabolite, it seems unlikely that finasteride has proconvulsant or other activity apart from enzyme inhibition that accounts for its interference with the anticonvulsant effects of progesterone. Moreover, by itself finasteride failed to affect the convulsant threshold of PTZ (Fig. 6). Thus, our results provide strong evidence that the anticonvulsant effect of progesterone in the PTZ test is due to a metabolite produced by 5α-reductase. Allopregnanolone is the presumed major GABAA receptor active 5α-reduced metabolite of progesterone and we assume that this steroid mediates the anticonvulsant activity of progesterone. However, until allopregnanolone levels are measured directly, this conclusion must remain tentative. Indeed, another GABAAreceptor-active steroid, allotetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one), could be synthesized via deoxycorticosterone (DOC), which is well recognized as a metabolite of progesterone (Winkel et al., 1980; Schneider and Honour, 1992). Following conversion from progesterone, DOC would then undergo the same sequential enzymatic conversions as proposed for progesterone in Fig. 1to form allotetrahydro-DOC (Kraulis et al., 1975). However, in comparison with the synthesis of allopregnanolone, the pathway through DOC to allotetrahydro-DOC is expected to be quantitatively minor.

Although the metabolic conversion of progesterone to allopregnanolone could occur in any of the numerous tissues known to contain finasteride-sensitive 5α-reductase isoenzymes (Li et al., 1995), it is of interest to note that brain is a rich source of the enzymes (Barnea et al., 1990; Melcangi et al., 1994). Brain also contains 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductases (Li et al., 1997), which are required for conversion of 5α-reduced progesterone (5α-dihydroprogesterone) to allopregnanolone (Fig. 1). The 5β-isomer of allopregnanolone (“pregnanolone”), like allopregnanolone itself, is a potent positive modulator of GABAA receptors (Harrison et al., 1987; Gee et al., 1988; Peters et al., 1988; Kokate et al., 1994). Although it has been proposed that pregnanolone can be synthesized from progesterone, evidence of a progesterone 5β-reductase activity has not been forthcoming (Kondo et al., 1994). Indeed, our results suggest that 5α-reduction is the main route whereby progesterone is activated, at least to produce its anticonvulsant activity.

Steroid 5α-reductase exists in two isoforms, designated as types I and II. The type I isoenzyme is more prominent in brain, whereas type II exists mainly in androgen-sensitive glandular tissues such as prostate (Celotti et al., 1997). In human tissues, finasteride inhibits both isoforms but is more potent at the type II than the type I isoenzyme, thus conferring prostatic selectivity (Stoner, 1990;Rittmaster, 1997). However, in the rodent finasteride is also a highly potent inhibitor of the type I enzyme (Thigpen and Russell, 1992;Azzolina et al., 1997). Because finasteride does not select between the rodent isoenzymes, our studies do not permit an assessment of the relative importance of the two isoenzymes in the metabolism of progesterone to allopregnanolone. In the future, it will be of interest to examine the activity of rodent isoenzyme selective 5α-reductase inhibitors.

The anticonvulsant activity of progesterone was originally demonstrated using an electroshock model (Spiegel and Wycis, 1945). In the present study, we also found progesterone to be active against electrically induced convulsions. However, protection in the MES activity only occurred at very high doses and, interestingly, was not prevented by finasteride (although high doses of finasteride itself had modest protective activity). Assuming that finasteride is able to fully block conversion of these high doses of progesterone, it can be concluded that the anticonvulsant mechanism of progesterone in the MES test is different from that in the PTZ test. GABAAreceptor potentiating agents are often weak or ineffective against MES seizures (Rogawski, 1996). Therefore, if the mechanism of progesterone’s anticonvulsant activity in the PTZ test is via a GABAA receptor potentiating neuroactive steroid, it is not surprising that the steroid has low potency in the MES test. The precise mechanism by which progesterone confers protection in this model remains to be determined. The lack of inhibitory effect of finasteride suggests that metabolic conversion by 5α-reduction is not required. It will be of interest to determine whether the effect of progesterone in the MES test occurs via interactions with ion channels other than GABAA receptors. Whether allopregnanolone would share this mechanism is not clear. Allopregnanolone produced only partial protection in the MES test. However, due to limited solubility, only doses as high as 100 mg/kg could be evaluated (see Kokate et al., 1994).

In summary, our results provide strong evidence that the anticonvulsant activity of progesterone is dependent upon activation by 5α-reductase isoenzymes. The active species is presumed to be mainly allopregnanolone, although other neuroactive steroid metabolites could also play a role. Several studies have indicated that progesterone therapy may be useful in women with catamenial epilepsy where fluctuations in circulating progesterone during the menstrual cycle could account for cyclic variations in seizure control (Herzog, 1995;Rodriguez Macias, 1996). Our results suggest that variations in brain levels of allopregnanolone or other progesterone-derived neuroactive steroids could explain the seizure exacerbations in catamenial epilepsy. Furthermore, because neuroactive steroids are devoid of hormonal activity (Paul and Purdy, 1992), they may be preferable to progesterone in the treatment of catamenial epilepsy.